Yiwei Zhang, Yuqiu Lan, Rufeng Fan, Lei Feng, Guoliang Wang, Xinyuan Wu, Lulu Wen, Zhiqiang Duan, Yueyue Xia, Xudong Wang, Lingrui Zhang, Lu Zhou, Minjia Tan, Cangsong Liao, Xiaojie Lu

Journal of the American Chemical Society

DOI: 10.1021/jacs.5c14634

Abstract

DNA-encoded libraries (DELs) have emerged as an effective and efficient selection strategy for lead compound discovery in academia and industry over the past few decades. Despite recent advancements in this field, DEL remains limited by sensitive DNA-based constructs, particularly with low selection success rates resulting from the random selection of targets. Here, we describe a chemoenzymatic on-DNA reaction for DEL syntheses and develop a chemoproteomic-guided DEL selection platform. This platform, termed FF tags-biocatDEL, integrates DEL technology, chemoenzymatic synthesis, and fully functionalized (FF) chemical tags to match DELs with selection targets, even with limited information about ligandable hotspots. Using two diazirine-based FF indole probes, we comprehensively surveyed binding partners in cells and identified phosphoglycerate dehydrogenase (PHGDH) as a potential target for DEL selection. DEL01 and DEL02 were designed, synthesized, and selected against PHGDH, leading to the identification of a novel enzyme-active compound with an IC₅₀ value of 2.5 μM. Our strategy, utilizing FF tags-biocatDEL, establishes a generalizable workflow for rapid target hunting and ligand discovery. It provides an effective method for precisely matching DELs with potential targets, demonstrating its significant potential as a complementary approach to drug discovery based on DELs.

Summary

This study presents a novel FF tags-biocatDEL platform that integrates chemoenzymatic synthesis, chemoproteomics, and DNA-encoded library (DEL) technology to overcome the low success rates of traditional DEL selection. The researchers developed a DNA-compatible decarboxylative aldol reaction using the PLP-dependent enzyme ApUstD to generate indole scaffolds bearing amine and carboxyl functional groups. Through chemoproteomic profiling with diazirine-based fully functionalized (FF) indole probes, they identified phosphoglycerate dehydrogenase (PHGDH) as a high-priority target from 2,208 enriched proteins. Two focused DELs were constructed: DEL01 (281,158 members via 2-cycle synthesis) and DEL02 (1.35 million members derived from a lactone fragment). Affinity selection against PHGDH yielded L5, a novel indole-based inhibitor with an IC₅₀ of 2.5 μM that acts via an allosteric mechanism. This strategy demonstrates that chemoproteomic guidance significantly enhances DEL selection efficiency and expands the chemical space for challenging targets.

Highlights

• Innovative Chemoenzymatic Reaction: The first application of ApUstD on DNA substrates, achieving quantitative conversion (up to 100%) under mild aqueous conditions to generate complex indole scaffolds with γ-hydroxy-α-amino acid structures.
• Chemoproteomic-Guided Target Identification: Diazirine-based FF indole probes enabled unbiased profiling of 2,208 ligandable proteins, with PHGDH emerging as a clinically relevant target (ranked 111th) for cancer and neurodegenerative diseases.
• Potent Allosteric Inhibitor Discovery: L5, derived from a lactone byproduct (L3) scaffold, showed sub-micromolar potency (IC₅₀ = 2.5 μM) and allosteric inhibition independent of NAD⁺ concentration, representing a new chemotype for PHGDH.
• Scaffold Optimization via DEL Iteration: Initial hits (L1, L2) showed modest activity, but leveraging a side-product scaffold (L3) to build DEL02 (1.35M compounds) enabled a 20-fold activity improvement over the parent fragment.
• Technical Milestones: Successfully synthesized large-scale DELs using biocatalysis, validated target engagement via photo-crosslinking and thermal shift assays, and established a generalizable workflow combining fragment-based DELs with proteome-wide targeting data.

Conclusion

The FF tags-biocatDEL platform successfully bridges biocatalysis, chemoproteomics, and DEL technology to create a highly efficient, target-directed drug discovery workflow. By using chemoproteomic data to rationally select PHGDH and focused DELs to optimize a biocatalytically derived indole scaffold, the team discovered L5, a novel, compact PHGDH inhibitor with promising activity. This approach significantly outperforms random target selection and expands the accessible chemical space for traditionally challenging enzymes. While the platform currently leverages biocatalysis primarily for scaffold generation, future expansion to multiple DEL synthesis steps could further enhance diversity. Additionally, the affinity-based selection may identify non-functional binders that could be repurposed as PROTACs or other modalities. Overall, this strategy offers a robust complement to conventional DEL methods and holds substantial promise for accelerating lead discovery against emerging therapeutic targets.